Bacterial meningitis continues to be an important cause of mortality and morbidity, such as developmental delay, hearing loss and cognitive impairment. The Metropolitan Atlanta Developmental Disabilities Surveillance Program identified bacterial meningitis as the leading postnatal cause of developmental disabilities. A major contributing factor to such mortality and morbidity is our incomplete knowledge on the pathogenesis of this disease. E. coli is the most common Gram-negative bacillary organism causing meningitis. Several lines of evidence from human cases and experimental animal models of E. coli meningitis indicate that E. coli penetration into the brain follows a high level f bacteremia and cerebral capillaries are the portal of E. coli entry into the brain. Since E. coli entry into the brain occurred in the cerebral microvessels, we developed the blood-brain barrier model with human brain microvascular endothelial cells (HBMEC) to study E. coli penetration of the blood-brain barrier. Our HBMEC monolayer, upon cultivation on collagen-coated Transwells, exhibits spatial organization of tight and adherens junction proteins as well as a polarized monolayer, a unique property of the blood-brain barrier endothelial cells. We have shown for the first time that meningitis-causing E. coli strains exhibit the ability to invade the HBMEC monolayer and that the ability of HBMEC invasion is correlated with E. coli penetration into the brain in vivo. The underlying mechanisms involved in E. coli penetration of the blood-brain barrier, however, remain incompletely understood. These findings indicate that new approaches are needed to investigate E. coli penetration of the blood-brain barrier. We have used E. coli invasion of HBMEC monolayer as a biologically relevant new approach for discovery of targets affecting E. coli penetration of the blood-brain barrier. This application is to take advantage of the new targets identified from our chemical screen for investigating how they contribute to E. coli penetration of the blood-brain barrier in vitro and in vivo. Characterization of such targets will demonstrate a novel strategy exploited by meningitis-causing E. coli strains for penetration of the blood-brain barrier, the essential step in the development of E. coli meningitis.